Introduction to countercurrent chromatography: instruments

These slides briefly explain the separation principle and the type of instruments utilized in countercurrent chromatography/ separation.
This workshop presentation was prepared by Dr. Friesen (http://www.dom.edu/departments/physicalsciences/faculty/j-brent-friesen).

5.
Countercurrent Distribution
Series of mixed and equilibrated individual cells
All cells contain stationary phase (lower phase in CCD instruments) at the beginning.
The first cell is mixed and allowed to settle.
The mobile phase is passed to the next cell in the series.
New mobile phase is added to the first cell.
The first cell contains an equal amount of upper and lower phase
plus the sample at the beginning.

8.
Countercurrent Distribution
Lyman C. Craig
Sequential countercurrent extractions can separate solutes
with only small differences in K. However, the technique, if
performed with separatory funnels, is quite tedious.
In 1944 Lyman C. Craig developed a device to automate
countercurrent distribution. This device used a series of
glass vessels.
8
http://onlinelibrary.wiley.com/doi/10.1111/j.1749-6632.1951.tb48879.x/pdf

11.
CounterCurrent Distribution
CCD equations:
The rate of migration of the band of a homogeneous substance, that is the position of
its maximum, can be calculated from Equation 1 at any time during operation, after a
certain number of plates, n, have been applied
IDENTIFICATION OF SMALL AMOUNTS OF ORGANIC COMPOUNDS BY DISTRIBUTION
STUDIES III. THE USE OF BUFFERS IN COUNTER-CURRENT DISTRIBUTION.
BY LYMAN C. CRAIG, CALVIN GOLUMBIC, HAROLD MIGHTON, AND ELWOOD TITUS
Journal of Biological Chemistry, 1945, vol. 161, p. 321

23.
Continuous Countercurrent
Separation in a centrifuge
Centrifuge is used to hold
one phase stationary.
Countercurrent Chromatography
http://pubs.acs.org/subscribe/journals/tcaw/10/i07/html/07inst.html
under the
influence of
centrifugal force
CounterCurrent Chromatography
through the planetary motion of
coils (bobbins)

34.
Determination of Sf
• (i) the carry-over method Sf(CO): this approach measures, in a
graduated cylinder, the amount of stationary phase carried over as
the column is equilibrated with the mobile phase. The amount of
stationary phase displaced is called the “carry over volume” or V(CO).
In the case of large volume columns, V(CO) is considered to be equal
to the mobile phase volume inside the column (VM).
• (ii) void volume determination by UV detection Sf(UV): this method is
routinely employed with crude natural extracts, is to identify the
mobile phase front by unretained UV-active sample components
that are almost always present in complex natural mixtures. The
void volume (V(UV)) is determined by taking the analyte retention
volume (VR) of an unretained component(s) (VR
0) to be equal to VM.
• (iii) volumetrics of extruded mobile phase Sf(MP): the third method is
based on the determination of VM by measuring the volume of
mobile phase extruded (V(MP)) from the column after the separation
is complete.
Pauli GF, Pro S, Chadwick L, Burdick T, Pro L, Friedl W, Novak, N, Maltby J, Qiu, F, Friesen JB
Real-Time Volumetric Phase Monitoring Advances Chemical Analysis by Countercurrent Separation Analytical Chemistry 87:7418-7425 (2015)
dx.doi.org/10.1021/acs.analchem.5b01613